Cell multiplication can be enhanced in kidney epithelial cells through the stimulation and release of endogenous growth factors. Growth factors comprise any substance, either genetic or extrinsic, which affect growth. Blakiston's Gould Medical Dictionary, 4th Edition, McGraw Hill Publishing Company, Copyrighted 1956, 1972 and 1979. There is interest in methods that increase cell replication through endogenous growth factors that include: one, the ability to recruit unstimulated cells to replicate; two, the enhancement of mitogenesis; and three, the regulation of ion and nutrient transport in adjacent and nonadjacent epithelial cells.
The self-stimulation of cell proliferation by endogenous growth factors was first demonstrated by Todaro and DeLarco in cultures of virus-transformed fibroblasts ("Growth factors produced by sarcoma virus-transformed cells" Cancer Res: 38: 4147-4154, 1978.). It was proposed that proliferation of these cells was under autocrine control since they secreted growth-promoting polypeptides. Further studies revealed that endogenous growth-stimulating proteins could be isolated from nontransformed fibroblasts and normal tissues. Thus, proliferation of fibroblasts could be mediated by the interaction of growth-stimulating and inhibiting polypeptides.
Later studies showed that the stimulation of renal epithelial cell growth could be induced by lowering the potassium concentration in extracellular fluid. (see "Growth of kidney epithelial cells in culture: evidence for autocrine control"; Lawrence J. Mordan and F. Gary Toback, Am. J. Physiol. 246: C351-C354, 1984). Specifically, the stimulation of renal epithelial cell growth, associated with the appearance of mitogenic factors in the extracellular fluid, was induced by lowering the extracellular potassium concentration. It was found that these renal epithelial cells transduce the information inherent in a reduction of the medium potassium concentration into endogenous growth-stimulating factors, which appeared in the medium.
Specifically, the study "Growth of kidney epithelial cells in culture: evidence for autocrine control" involved exposing epithelial cells from the African green monkey kidney (BSC-1 line) to three different types of media: one, low potassium conditioned medium; two, control conditioned medium; and three, unconditioned fresh medium. The study revealed that the epithelial cells that were exposed to fresh low potassium medium (3.2 millimolar potassium) grew to a higher density than cells in control medium (5.4 millimolar potassium). Low potassium medium, which had been conditioned by cultures of confluent quiescent BSC-1 cells for at least 1 hour, was collected and adjusted to 5.4 millimolar potassium by adding potassium chloride. The adjusted conditioned low potassium medium was then applied to fresh cultures to assess its effect on cell growth.
The results showed that sparse cultures of BSC-1 cells exposed to this medium grew to a higher density than cultures exposed to conditioned medium prepared at the control potassium concentration. Growth-stimulating activity in low potassium conditioned medium was optimal at a potassium concentration of 3.2 millimolar, which was the same potassium concentration required for optimal growth with unconditioned fresh medium. One hour of exposure of BSC-1 cells to low potassium conditioned medium was required to increase the growth-stimulating activity of this medium above that in control conditioned medium. This amount of growth-stimulating activity was constant for up to 12 hours. Control medium conditioned for 12 hours enhanced growth compared with media conditioned for less time. Both control and low potassium conditioned media stimulated growth to a greater extent after 15 to 45 minutes than did unconditioned fresh medium.
Thus, this study indicated that growth-stimulating activity in low-potassium conditioned medium was optimal when the potassium concentration during the one to six hours of conditioning was 3.2 millimolar. Control conditioned medium also contained growth-stimulating activity compared with unconditioned medium. Hence, the results suggested that BSC-1 cells exposed to low-potassium medium produce a larger quantity of growth-stimulating factors and/or different ones than do control cells.
Prior to the present invention, it was known that renal epithelial cell growth could be stimulated by lowering the concentration of extracellular potassium. However, alternatives to that state of the art are important, especially if lowering the extracellular potassium concentration is not desired. The present invention provides an alternative to lowering the concentration of extracellular potassium to produce autocrine growth factors.